Field of the Invention
The present invention relates in general to diagnostic assays for detecting protease activity associated with cancer, correlation of diagnostic results with cancer prognosis, and methods of detecting the presence of a cancerous or precancerous cell.
Description of the Prior Art
Cancer prognosis is based upon the stage of the disease. The four major stages are initial mutations, cell survival and tumor progression, angiogenesis, and finally invasion or metastasis. One of the biggest challenges is that cancer is a disease of the body's own cells. Because of this, it is often very challenging to diagnose cancer until the disease is quite progressed. This also affects treatment efficiency, since the cancer cells have the same enzymes, replication equipment, structural features, etc. as healthy cells, treatments that delineate cancer cells from healthy cells are difficult to develop. This is why staging and treatment is usually based on the symptoms (i.e., the size of the tumor, whether lymph nodes contain cancer, and whether the cancer has spread from the original site to other parts of the body), rather than on specifically targeting the origin of the cancer.
More recently, a number of proteases have been associated with disease progression in cancer. Proteases are a class of enzyme that catalyze the cleavage of the peptide bond in other proteins. They can be very specific (only being able to degrade one peptide bond in one protein) or extremely broad (e.g., being able to cleave the peptide bond every time there is, for example, a lysine). Several proteases are known to be over-expressed by various cancer cell lines. Proteases that are known to be necessary for cancer development and progression include Matrix Metalloproteinases (MMPs), Tissue Serine Proteases, and the Cathepsins. Many of these proteases are either upregulated in the cancer cells (that is, they have a much higher activity in the tumor than in healthy tissue), mis-expressed (that is, they are found in compartments where they should not be found), or are involved in embryonic development, but should not be found to any significant extent in an adult cells.
MMPs are the classic cancer-associated proteases. MMPs are a family of zinc proteases that are named for the zinc and calcium ions that are required as cofactors. There are 21 different known MMPs that are grouped into families based on their substrates: collagenases, gelatinases, stromelysins, matrilysin, metalloelastase, enamelysin, and membrane-type MMPs. As can be seen from the family names, MMPs degrade the proteins that make up the extracellular matrix (ECM) and the basement membrane (BM) of tissue. MMPs are usually not produced by the cancerous cells themselves, but by the stromal cells surrounding the tumor. This is because the cancerous cells give off a variety of cell signals that cause the surrounding stromal cells to highly upregulate their production of MMPs. MMPs are vital to cancer survival and progression for several reasons. First, they cleave cell surface bound growth factors from the stromal and epithelial cells and release them to interact with the cancer cells to stimulate growth. They also play a role in angiogenesis by opening the ECM to new vessel development as well as by releasing pro-angiogenic factors and starting pro-angiogenic protease cascades. MMPs play a major role in tumor metastasis by degrading the ECM and the BM, allowing the cells to pass through tissue barriers. They also release ECM and BM fragments, which stimulates cell movement.
Several serine proteases have well-documented roles in cancer as well, especially urokinase plasminogen activator (uPA) and plasmin. Elevated expression levels of urokinase and several other components of the plasminogen activation system have been found to be correlated with tumor malignancy. uPA is a very specific protease that binds to its receptor, uPAR, and cleaves the inactive plasminogen (a zymogen) to the active plasmin. This is the first step in a well-known cascade that causes angiogenesis. It is believed that the tissue degradation that follows plasminogen activation facilitates tissue invasion and contributes to metastasis.
Cathepsins, with a few exceptions, are cysteine proteases. Often found in the lysosomal/endosomal pathway, cathepsins usually operate at low pH values, but some are still active at neutral pH. Three of the cathepsins, B, D, and L, are active at neutral pH and are often misexpressed in cancer, causing activation outside of the cells. This activation outside of the cell can cause ECM degradation.
Many studies directed to assess the prognostic impact of the plasminogen/plasmin components have been conducted, mostly based on antigen level quantitation in tissue extracts from surgically removed tumors. These values have been subsequently correlated with prognosis in several types of cancers. However, determining ways to distinguish cancerous cells from healthy cells remains a large area of research in cancer therapy, and there is still a need in the art for methods of quantitatively detecting cancer progression and stages of the disease that can be applied in vitro and in vivo. There is also a need for detecting multiple markers of the various cancer stages at one time without the need for separate tests. There also is a need for in vivo characterization of cancer, so that treatment can be directed to the most malignant cancer tissue.